Methods of using small rna from bodily fluids for diagnosis and monitoring of neurodegenerative diseases

ABSTRACT

Described are methods for detection of neuronal pathologies using quantitative analysis in bodily fluids of synapse and/or neurite small RNAs and application of these methods to early diagnosis and monitoring of neurodegenerative diseases and other neurological disorders.

TECHNICAL FIELD OF THE INVENTION

The present invention is directed to methods for noninvasive orminimally invasive detection of pathological changes in brain or otherneurons by quantifying neurite and/or synapse small RNA, particularlymiRNA, in bodily fluids and application of these methods to earlydiagnosis and monitoring of neurodegenerative diseases and otherneurological disorders.

BACKGROUND OF THE INVENTION

Neurodegenerative diseases comprise a large group of pathologies causedby metabolic changes in brain cells, loss of synapses and othercompartments of neurons, and finally neuronal death. For review seeNeurodegenerative diseases: From Molecular Concepts to TherapeuticTargets. Editors: R. von Bernhardi, N.C. Inestrosa, Nova Publishers,2008. This group of diseases includes Mild Cognitive Impairment (MCI),Alzheimer's disease (AD), Lewy Body dementia, Parkinson's disease (PD),Huntington's disease (HD), frontotemporal dementia (FTD), vasculardementia, HIV Associated Neurocognitive Disorders (HAND), multiplesclerosis (MS), amyotrophic lateral sclerosis (ALS), prion diseases,different ataxias, and others. Due to increased lifespan,neurodegenerative diseases have become very common in developedcountries. There are about 6 million people living with AD in the USonly, 70-80 million people are in the risk group and $148 billion isspent in the US for AD patient treatment and care. Drug development andsuccessful treatment of AD and other neurodegenerative diseases aresignificantly complicated by the absence of effective methods for theirearly diagnosis and monitoring. Development of effective diagnosticmethods is further complicated by the strong brain potential tocompensate for the dysfunction and loss of neurons over a long period oftime. This results in late clinical manifestation of disease symptomswhen treatment cannot be very successful due to serious morphologicchanges in the brain including the massive loss of neurons. Thus,diagnostic methods based on detection of early events in the diseasedevelopment are particularly desirable.

Neurodegenerative diseases are characterized by neuronal death indifferent disease-specific areas of the brain. However, the neuronalloss is a relatively late event, typically following synapticdysfunction, synaptic loss, neurite retraction, and the appearance ofother abnormalities such as axonal transport defects. See, e.g.,Bredesen, Molecular Neurodegeneration 2009, 4:27; Siskova et al., Am JPathol. 2009, 175(4):1610-21; Kielar et al., Hum Mol Genet. 2009,18(21):4066-4080; Nimmrich and Ebert, Rev Neurosci. 2009, 20:1-12;Bellizzi et al., J Neuroimmune Pharmacol. 2006, 1:20-31; Milnerwood andRaymond, J Physiol. 2007, 585:817-831; Waataja et al., J Neurochem.2008, 104:364-375; Fuhrmann et al., J Neurosci. 2007, 27:6224-6233;Yoshiyama et al., Neuron. 2007, 53:337-351; Wishart et al., JNeuropathol Exp Neurol. 2006, 65:733-739; Gylys et al., Neurochem Int.2004; 44:125-131; Conforti et al., Trends Neurosci. 2007, 30:159-166;Baloyannis et al., J Neurol Sci. 2006, 248:35-41; Diaz-Hernandez et al.,FASEB J. 2009, 23:1893-1906; Spampanato et al., Neuroscience 2008,157:606-620; Wade et al., Brain Res. 2008, 1188:61-68; Centonze et al.,J Neurosci. 2009, 29:3442-3452; Wegner et al., Neurology. 2006,67:960-967; Dupuis and Loeffler, Cliff Opin Pharmacol. 2009, 9:341-346;Revuelta, et al. Am J Alzheimers Dis Other Demen 2008 23: 97-102.Numerous studies are devoted to description of axon destruction withshedding of membrane-enclosed “axosomes”, axon, dendrite and spinepruning, and disassembly of synapses (Goda, Davis, Neuron 2003,40:243-264; Eaton, Davis, Genes Development, 2003, 17:2075-2082; Koiral,Ko, Neuron, 2004, 44:578-580; Bishop et al., Neuron, 2004, 44:651-661;Low, Cheng, Phil. Trans. R. Soc. B 2006 361, 1531-1544).

Currently, diagnosis of AD and other forms of dementia is based onanalysis of the patient's cognitive function. As mentioned above, due toeffective compensatory mechanisms in the brain, the decrease ofcognitive function is usually registered when a disease is in its laterstages and fewer treatments are available. New imaging techniques, whichare becoming increasingly popular (e.g., positron emission tomography(PET), computed tomography (CT), magnetic resonance imaging (MRI),multiphoton imaging, magnetoencephalography (MEG),electroencephalography (EEG) etc.), are helpful, however, they arecurrently not sufficiently sensitive and specific for detecting earlystages of a disease before major morphological changes occur (Mucke,Nature, 2009, 461:895-897; Mistur et al., J. Clin. Neurol., 2009,5:153-166; Miller, Science, 2009, 326:386-389; Perrin et al., Nature,2009, 461: 916-922).

The existing diagnostic molecular tests for AD and other forms ofdementia can be divided into two groups. The first group is based onanalysis of single nucleotide polymorphisms (SNP), which is helpful forpredicting a higher risk of a disease but not for diagnostics (Bettenset al., Hum Mol Genet. 2010, 19(R1):R4-R11). The second group usesanalysis of proteins involved in AD pathogenesis or brain-specificproteins, like neural thread protein (NTP), in bodily fluids (Schipper,Alzheimer's & Dementia. 2007, 3:325-332). However, these tests are notsufficiently sensitive and specific. Recently published data havedemonstrated high sensitivity of AD detection by measuringconcentrations of three protein biomarkers (beta-amyloid protein 1-42,total tau protein, and phosphorylated tau181P protein) in thecerebrospinal fluid (CST) (Meyer et al., Arch Neurol. 2010, 67:949-956).The high invasiveness of the CSF collection procedure makes such testsimpractical and challenging for everyday clinical use.

Metabolic changes occurring in neurodegenerative diseases cause thedestruction of spines, dendrites, axons, and synapse loss, and thelatter, most likely, induces neuronal death (Bredesen, MolecularNeurodegeneration 2009, 4:27). Similar processes happen during embryonicbrain development. Numerous neurons are trying to establishintercellular contacts, those neurons that do it successfully survive,and other neurons die (Butts et al., Cell Death Differ. 2008,15:1178-1186; Enokido and Hatanaka, Gan To Kagaku Ryoho. 1994,21:615-620; Gasic and Nicotera, Toxicol Lett. 2003, 139:221-227).

Axon destruction with shedding of membrane-enclosed “axosomcs”, axon,dendrite and spine pruning, and disassembly of synapses lead toappearance of cell-free vesicles containing cytoplasmic components ofneurons, axons, neurites, spines and synapses, including proteins, RNAand their degradation products. There are other processes leading toliberation of these compounds into the extracellular medium, inparticular, blebbing (Charras et al., Biophys. J. 2008, 94:1836-1853;Fackler, Grosse, J. Cell Biol. 2008, 181:879-884), exocytosis (Skog etal. Nat Cell Biol., 2008, 10:1470-1476) and other forms of activesecretion.

MicroRNAs (miRNAs) are a class of non-coding RNAs whose final product isan approximately 22 nt functional RNA molecule. They play importantroles in the regulation of target genes by binding to complementaryregions of messenger transcripts to repress their translation orregulate degradation (Griffiths-Jones Nucleic Acids Research, 2006, 34,Database issue: D140-D144). Frequently, one miRNA can target multiplemRNAs and one mRNA can be regulated by multiple miRNAs targetingdifferent regions of the 3′ UTR. Once bound to an mRNA, miRNA canmodulate gene expression and protein production by affecting, e.g., mRNAtranslation and stability (e.g., Baek et al., Nature 455(7209):64(2008); Selbach et al., Nature 455(7209):58 (2008); Ambros, 2004,Nature, 431, 350-355; Bartel, 2004, Cell, 116, 281-297; Cullen, 2004,Virus Research., 102, 3-9; He et al., 2004, Nat. Rev. Genet., 5,522-531; and Ying et al., 2004, Gene, 342, 25-28). There are otherclasses of less characterized small RNAs (reviewed in Kim, Mol. Cells,2005, 19: 1-15).

Many of miRNAs are specific to or over-expressed in certainorgans/tissues/cells. See, e.g., Hua et al., BMC Genomics 2009, 10:214;Liang et al., BMC Genomics. 2007, 8:166; Landgraf et al., Cell. 2007,129:1401-1414; Lee et al., RNA. 2008, 14:35-42.

Some miRNAs, including those that are cell-specific, are enriched incertain cellular compartments, particularly in axons, dendrites andsynapses. See, e.g., Schratt et al., Nature. 439:283-289, 2006; Lugli etal., J Neurochem. 106:650-661, 2008; Bicker and Schratt, J Cell MolMed., 12:1466-1476, 2008; Smalheiser and Lugli, Neuromolecular Med.11:133-140, 2009; Rajasethupathy, Neuron. 63:714-716, 2009; Kye, RNA13:1224-1234, 2007; Yu et al., Exp Cell Res. 314:2618-2633, 2008;Cougot, et al., J Neurosci. 28:13793-13804, 2008; Kawahara, Brain Nerve.60:1437-1444, 2008; Schratt G. Rev Neurosci. 2009; 10:842-849.

Expression and concentrations of miRNAs are regulated by variousphysiological and pathological signals. Changes in expression of somemiRNAs were found in neurons of Alzheimer's and other neurodegenerativedisease patients. Hébert and Dc Strooper, Trends Neurosci. 32:199-206,2009; Saba et al., PLoS One. 2008; 3:e3652; Kocerha et al.,Neuromolecular Med. 2009; 11:162-172; Sethi and Lukiw, Neurosci Lett.2009, 459:100-104; Zeng, Mol Pharmacol. 75:259-264, 2009; Cogswell etal., Journal of Alzheimer's Disease. 14: 27-41, 2008; Schaefer et al.,J. Exp. Med. 204:1553-1558, 2007; Hébert, Proc Natl Acad Sci USA. 2008;105:6415-6420; Wang et al., J Neurosci. 2008, 28:1213-1223; Nelson etal., Brain Pathol. 2008; 18:130-138; Lukiw, Neuroreport. 2007;18:297-300.

Due to their small size, miRNAs can cross the blood-brain, placental andkidney barriers. Analysis of cell/tissue-specific miRNAs in bodilyfluids was proposed for detection of in vivo cell death (U.S. PatentPub. No 20090081640; Laterza et al., Clin Chem. 2009, 55:1977-1983).

Cognitive function testing and brain imaging, which are currently usedas main methods for diagnosis of neurodegenerative diseases such as AD,allow only detection of later stages of disease and are not sufficientlyspecific. There is still a great need in the art to develop methods forearly diagnosis of neurodegenerative diseases and other neurologicaldisorders in mammals prior to occurrence of major morphological changesand massive neuronal cell death.

SUMMARY OF THE INVENTION

The present invention addresses these and other needs by providing anovel highly sensitive and noninvasive or minimally invasive diagnosticand monitoring methods based on quantification in bodily fluids ofsynapse and/or neurite small RNAs. The methods of the present inventionallow diagnosis and monitoring of neurodegenerative diseases and otherneurological disorders prior to occurrence of major morphologicalchanges and massive neuronal cell death and thus have numerous clinicalimplications. For example, the use of the methods of the presentinvention can lead to enhanced effectiveness of currently availabletreatments for neurodegenerative diseases and other neurologicaldisorders as such treatments could be administered at a significantlyearlier stage of the disease. The use of the methods of the presentinvention can also allow development of new effective therapeutic and/orpreventive treatments and can decrease costs and increase efficiency ofclinical trials associated with such development.

In the first object, the present invention provides a method fordiagnosing a neuronal pathology in a subject, which comprises:

-   -   a. determining the level of at least one synapse and/or neurite        small RNA in a bodily fluid sample from the subject;    -   b. comparing the level of the small RNA in the bodily fluid        sample from the subject with a control level of the small RNA,        and    -   c. (i) identifying the subject as being afflicted with the        neuronal pathology when the level of the small RNA in the bodily        fluid sample from the subject is increased as compared to the        control or (ii) identifying the subject as not being afflicted        with the neuronal pathology when the level of the small RNA in        the bodily fluid sample from the subject is not increased as        compared to the control.

In another aspect, the invention provides a method for diagnosing aneuronal pathology in a subject, which comprises:

-   -   a. determining the level of a synapse and/or neurite small RNA        in a bodily fluid sample from the subject;    -   b. determining the level of a neuronal body small RNA (e.g.,        miR-181a or miR-491-5p) in a bodily fluid sample from the        subject;    -   c. determining the ratio of the levels of the small RNAs        determined in steps (a) and (b);    -   d. comparing the ratio of the levels of the small RNAs        determined in step (c) with a corresponding control ratio, and    -   e. (i) identifying the subject as being afflicted with the        neuronal pathology when the ratio of the levels of the small        RNAs determined in step (c) is higher than the corresponding        control ratio or (ii) identifying the subject as not being        afflicted with the neuronal pathology when ratio of the levels        of the small RNAs determined in step (c) is not higher than the        corresponding control ratio.

In a related aspect, the invention provides a method for diagnosingAlzheimer's disease (AD) in a subject, which comprises:

-   -   a. determining the level of at least one synapse or neurite        small RNA in a bodily fluid sample from the subject;    -   b. comparing the level of the small RNA in the bodily fluid        sample from the subject with a control level of the small RNA,        and    -   c. (i) identifying the subject as being afflicted with AD when        the level of the small RNA in the bodily fluid sample from the        subject is increased as compared to the control or (ii)        identifying the subject as not being afflicted with AD when the        level of the small RNA in the bodily fluid sample from the        subject is not increased as compared to the control.

In another related aspect, the invention provides a method fordiagnosing mild cognitive impairment (MCI) in a subject, whichcomprises:

-   -   a. determining the level of at least one synapse or neurite        small RNA in a bodily fluid sample from the subject;    -   b. comparing the level of the small RNA in the bodily fluid        sample from the subject with a control level of the small RNA,        and    -   c. (i) identifying the subject as being afflicted with MCI when        the level of the small RNA in the bodily fluid sample from the        subject is increased as compared to the control or (ii)        identifying the subject as not being afflicted with MCI when the        level of the small RNA in the bodily fluid sample from the        subject is not increased as compared to the control.

In any of the above diagnostic methods, the control level of the smallRNA can be, for example, (i) the level of said small RNA in a similarlyprocessed bodily fluid sample from an age-matched control subject, (ii)the level of said small RNA in a similarly processed bodily fluid samplefrom the same subject obtained in the past, or (iii) a predeterminedstandard.

Any of the above diagnostic methods can further comprise normalizing thelevel of the small RNA in the bodily fluid sample from the subject andin the control to the level of a small RNA which is not expressed inbrain (e.g., miR-10b or miR-141).

In another aspect, the invention provides a method for monitoringdevelopment of a neuronal pathology in a subject, which comprises:

-   -   a. determining the level of at least one synapse or neurite        small RNA in two or more bodily fluid samples from the subject,        wherein the samples have been obtained at spaced apart time        points (e.g., within 1-48 months intervals), and    -   b. comparing the levels of the small RNA between the earlier        obtained and later obtained bodily fluid sample(s).

Such method can further comprise (c) (i) determining that thedevelopment of the neuronal pathology in the subject is accelerated ifthe level of the small RNA is increased in the later obtained bodilyfluid sample(s) as compared to the earlier obtained sample(s); (ii)determining that the neuronal pathology in the subject continues todevelop at the same rate if the level of the small RNA is not changed inthe later obtained bodily fluid sample(s) as compared to the earlierobtained sample(s), and (iii) determining that the development of theneuronal pathology in the subject is slowed down if the level of thesmall RNA is decreased in the later obtained bodily fluid sample(s) ascompared to the earlier obtained sample(s).

In an additional aspect, the invention provides a method for monitoringthe effectiveness of a treatment of a neuronal pathology in a subject,which comprises:

-   -   a. determining the level of at least one synapse or neurite        small RNA in a bodily fluid sample from the subject obtained        prior to initiation of the treatment;    -   b. determining the level of the small RNA in one or more bodily        fluid sample(s) from the subject obtained in the course of or        following the treatment (e.g., within 1 week-12 months        intervals), and    -   c. comparing the level of the small RNA determined in steps (a)        and (b), and optionally between different samples in step (b).

Such method can further comprise (d) (i) determining that the treatmentis effective if the level of the small RNA has decreased in the courseof or following the treatment or (ii) determining that the treatment isnot effective if the level of the small RNA has not decreased in thecourse of or following the treatment.

Non-limiting examples of neuronal pathologies which can be diagnosed andmonitored using any of the above methods include neurodegenerativediseases (such as, e.g., Alzheimer's disease (AD), Parkinson's disease(PD), Lewy Body dementia, Huntington's disease (HD), frontotemporaldementia (FTD), vascular dementia, HIV Associated NeurocognitiveDisorders (HAND), mild cognitive impairment (MCT), mixed dementia,Creutzfeldt-Jakob Disease (CJD), normal pressure hydrocephalus,Wernicke-Korsakoff syndrome, multiple sclerosis (MS), amyotrophiclateral sclerosis (ALS), prion diseases, and different ataxias) andneuronal pathologies associated with an encephalopathy or neuropathy.

In any of the above methods, a neuronal pathology can be diagnosed andmonitored prior to massive neuronal cell death characteristic of saidpathology.

In one embodiment, the small RNA used in any of the methods of theinvention is present in synapses. In another embodiment, the small RNAused in any of the methods of the invention is present in spines. In yetanother embodiment, the small RNA used in any of the methods of theinvention is present in axons. In a further embodiment, the small RNAused in any of the methods of the invention is present in dendrites.

In one embodiment, the small RNA used in any of the methods of theinvention is miRNA. Non-limiting examples of synapse and/or neuritemiRNAs useful in any of the methods of the invention include miR-7,miR-9, miR-9*, miR-25, miR-26a, miR-26b, miR-98, miR-124, miR-125b,miR-128, miR-132, miR-134, miR-138, miR-146, miR-182, miR-183, miR-200b,miR-200c, miR-213, miR-292-5p, miR-297, miR-322, miR-323-3p, miR-325,miR-337, miR-339, miR-345, miR-350, miR-351, miR-370, miR-425, miR-429,miR-433-5p, miR-446, miR-467, and miR-874. In one specific embodiment,the miRNA is selected from the group consisting of miR-7, miR-125b,miR-128, miR-132, miR-323-3p, miR-370, and miR-874.

In one embodiment, any of the methods of the invention comprisedetermining the level of two or more synapse and/or neurite small RNAs.

Non-limiting examples of bodily fluid samples useful in any of themethods of the invention include blood plasma, serum, urine, and saliva.

Non-limiting examples of methods for determining the level of small RNAsuseful in any of the methods of the invention include hybridization,RT-PCR, and sequencing.

In one embodiment, prior to step (a) in any of the above methods, thesmall RNA is purified from the bodily fluid sample.

Any of the above methods can further comprise the step of reducing oreliminating degradation of the small RNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-G are graphs showing comparisons of miRNA concentrations inplasma of AD patients and age-matched controls. All concentrations werenormalized per ubiquitous miR-16 and presented in relative units(ordinate axis). miR-7 (A), miR-125b (B), miR-128 (C), miR-132 (D), andmiR-323-3p (E) are neurite and/or synapse miRNA; miR-181a (F) andmiR-491-5p (G) are neuronal body miRNA.

FIGS. 2A-D are graphs showing comparison of miRNA concentrations inplasma of MCT patients and age-matched controls. All concentrations werenormalized per spiked miRNA and presented in relative units (ordinateaxis) miR-7 (A) and miR-874 (B) are neurite and/or synapse miRNA;miR-181a (C) and miR-491-5p (D) are neuronal body miRNA.

FIGS. 3A-B are graphs showing comparison of miRNA concentrations inplasma of MCI patients and age-matched controls. All concentrations werenormalized per miR-141 and presented in relative units (ordinate axis).miR-128 (A) is neurite and synapse miRNA; miR-539 (B) is neuronal bodymiRNA.

FIGS. 4A-D are graphs showing comparison of miRNA concentrations inplasma of MCI and AD patients and age-matched controls. Concentrationsof neurite and/or synapse miRNA miR-128 (A), miR-132 (B), miR-370 (C),and miR-125b (D) were normalized per miR-181a (neuronal body miRNA) andpresented in relative units (ordinate axis).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the inventors' realization that sinceneurite (axon and/or dendrite and/or spine) destruction and synapse lossas well as some metabolic events precede neuronal death in the course ofdevelopment of neurodegenerative diseases, methods based on detection ofthose phenomena could be used for earlier disease diagnosis than theones based on detecting cell death.

The instant invention is further based on the inventors' discovery thatlevels of synapse and/or neurite miRNAs increase in bodily fluids ofpatients with Mild Cognitive Impairment (MCI) and/or Alzheimer's disease(AD) compared to respective age-matched controls reflecting excessivedestruction of neurites and/or loss of synapses.

Within the meaning of the present invention, the term “synapse and/orneurite small RNA” refers to small RNA (e.g., miRNA or BC200 RNA) which(i) is “neuron-enriched”, i.e., is present in increased amounts (e.g.,at least 5-times higher concentrations) in neurons, as compared to celltypes that can be a source of significant amounts of small RNA in abodily fluid being tested and (ii) is present in a synapse and/orneurite (i.e., axon and/or dendrite and/or spine). To be useful in thediagnostic methods of the present invention, such synapse and/or neuritesmall RNA should be detectable in bodily fluids as a result of itsrelease from neurons (e.g., due to neurite/synapse destruction orneuronal death).

The present invention provides a novel highly sensitive and noninvasiveor minimally invasive method for diagnosing a neuronal pathology (e.g.,a neuronal pathology associated with a neurodegenerative disease oranother neurological disorder) in a subject, said method comprisingdetermining the level in a bodily fluid sample from the subject (e.g.,blood plasma or scrum, urine, saliva, or other bodily fluids) of one ormore synapse and/or neurite small RNA (e.g., miRNA or BC200 RNA).Specifically, this method comprises (a) determining the level of atleast one synapse and/or neurite small RNA in a bodily fluid sample fromthe subject; (b) comparing the level of the small RNA in the bodilyfluid sample with a control level of the small RNA (e.g., a similarlyprocessed bodily fluid sample from a control subject [e.g., anage-matched control or the same subject in the past (e.g., 1, 3, 6, 12,24, 36, or 48 months earlier)] or a predetermined standard), and (c) (i)identifying the subject as being afflicted with the neuronal pathologywhen the level of the small RNA in the bodily fluid sample from thesubject is increased as compared to the control or (ii) identifying thesubject as not being afflicted with the neuronal pathology when thelevel of the small RNA in the bodily fluid sample from the subject isnot increased as compared to the control.

The diagnostic method of the invention makes possible early diagnosis ofneurodegenerative diseases and other neurological disorders, e.g., priorto occurrence of major morphological changes and/or massive neuronalcell death associated with such diseases and disorders.

Furthermore, analysis of synapse and/or neurite small RNAs significantlyenhances the sensitivity of the small RNA detection as compared todetecting neuronal body small RNAs which are not present or depleted insynapses and neurites, because the amount of synapses and neurites inthe brain is 10³ times higher than the amount of neurons. This approachalso provides detailed and comprehensive information for monitoringdisease development and treatment effectiveness, since various specificevents in neurons (e.g., changes in miRNA profile, their secretion,neurite degradation, synapse loss, and finally neuronal death) can bedetected and quantitated.

Differences in levels of synapse and/or neurite small RNAs in bodilyfluids of subjects having neurodegenerative diseases or otherneurological disorders as compared to normal subjects detectable by themethod of the present invention may be due to (i) disease-associateddestruction of neurites and/or synapses, (ii) disease-associated changesin expression or metabolism of these small RNAs, (iii)disease-associated changes in transport and intracellular distributionof these small RNAs, (iv) disease-associated changes in secretion ofthese small RNAs (Rabinowits et al. Clin Lung Cancer, 2009, 10:42-46),as well as other causes.

In a separate embodiment, the invention provides a related diagnosticmethod for diagnosing a neuronal pathology which comprises (a)determining the level of a synapse and/or neurite small RNA in a bodilyfluid sample from the subject; (b) determining the level of a neuronalbody small RNA in a bodily fluid sample from the subject; (c)determining the ratio of the levels of the small RNAs determined insteps (a) and (b); (d) comparing the ratio of the levels of the smallRNAs determined in step (c) with a corresponding control ratio, and (e)(i) identifying the subject as being afflicted with the neuronalpathology when the ratio of the levels of the small RNAs determined instep (c) is higher than the corresponding control ratio or (ii)identifying the subject as not being afflicted with the neuronalpathology when ratio of the levels of the small RNAs determined in step(c) is not higher than the corresponding control ratio.

Within the meaning of the present invention, the term “neuronal bodysmall RNA” refers to small RNA (e.g., miRNA) which (i) is“neuron-enriched”, i.e., is present in increased amounts (e.g., at least5-times higher concentrations) in neurons, as compared to cell typesthat can be a source of significant amounts of small RNA in a bodilyfluid being tested and (ii) is absent from or present in significantlylower concentrations in neurites or synapses than in neuronal cellbodies.

In another related embodiment, the present invention provides a methodfor monitoring development of a neuronal pathology (e.g., a neuronalpathology associated with a neurodegenerative disease or anotherneurological disorder) by periodically (e.g., every 1, 3, 6, 12, 24, 36,48 months) obtaining samples of a bodily fluid from a subject underobservation and determining changes in the level of one or more synapseand/or neurite small RNA (e.g., miRNA or BC200 RNA) in the bodily fluid.Specifically, this method comprises (a) determining the level of atleast one synapse and/or neurite small RNA in two or more bodily fluidsamples from the subject, wherein the samples have been obtained atspaced apart time points, and (b) comparing the levels of the small RNAbetween the earlier obtained and later obtained bodily fluid sample(s).If the level of the small RNA is increased in the later obtained bodilyfluid sample(s) as compared to the earlier obtained sample(s), this isindicative of acceleration of development of the neuronal pathology inthe subject. If the level of the small RNA is not changed in the laterobtained bodily fluid sample(s) as compared to the earlier obtainedsample(s), this is indicative that the neuronal pathology in the subjectcontinues to develop at the same rate. If the level of the small RNA isdecreased in the later obtained bodily fluid sample(s) as compared tothe earlier obtained sample(s), this is indicative of slow down indevelopment of the neuronal pathology in the subject.

In another related embodiment, the invention provides a method formonitoring the effectiveness of a treatment of a neuronal pathology(e.g., a neuronal pathology associated with a neurodegenerative diseaseor another neurological disorder) in a subject by determining changes inthe level of one or more synapse and/or neurite small RNA in bodilyfluid samples from the subject, wherein said samples have been obtainedprior to initiation of the treatment and at different time points (e.g.,every 1 week, 2 weeks, 1 month, 3 months, 6 months, 12 months, 24months, 36 months, 48 months) in the course of or following thetreatment. Specifically, this method comprises (a) determining the levelof at least one synapse and/or neurite small RNA in a bodily fluidsample from the subject obtained prior to initiation of the treatment;(b) determining the level of the small RNA in one or more bodily fluidsample(s) from the subject obtained in the course of or following thetreatment, and (c) comparing the level of the small RNA determined insteps (a) and (b), and optionally between different samples in step (b).If the level of the small RNA has decreased in the course of orfollowing the treatment, this is indicative that the treatment iseffective. If the level of the small RNA has not decreased in the courseof or following the treatment, this is indicative that the treatment isnot effective. This method can also involve comparison with placebotreated patients or other relevant controls.

The diagnostic and monitoring methods of the invention are useful fordetecting and monitoring any stage of development of a neuronalpathology (e.g., a neuronal pathology associated with aneurodegenerative disease or another neurological disorder) and providethe advantage of a simple and minimally invasive (or noninvasive) assay.As noted above, unlike methods known in the art, the methods of theinvention allow for diagnosis and monitoring of neuronal pathologiesprior to occurrence of major morphological changes and/or massiveneuronal cell death associated with such pathologies.

The methods of the present invention can be used to diagnose and monitorvarious neuronal pathologies including, without limitation,neurodegenerative diseases (e.g., Alzheimer's disease (AD), Parkinson'sdisease (PD), Lewy Body dementia, Huntington's disease (HD),frontotemporal dementia (FTD), vascular dementia, HIV AssociatedNeurocognitive Disorders (HAND), mild cognitive impairment (MCI), mixeddementia, Creutzfeldt-Jakob Disease (CJD), normal pressurehydrocephalus, Wernicke-Korsakoff syndrome, multiple sclerosis (MS),amyotrophic lateral sclerosis (ALS), prion diseases, different ataxias,etc.), various encephalopaties (e.g, viral encephalopaties such as AIDSdementia) and neuropathies (e.g., glaucoma [optical neuropathy], spinalmuscular atrophy, etc.). In a separate embodiment of the presentinvention, a spectrum of various small RNAs (e.g., various miRNAs) canbe analyzed for differential diagnosis of various neurodegenerativediseases with similar clinical symptoms, for example, different forms ofdementia.

Neurite and/or synapse small RNAs useful in the methods of the presentinvention include, without limitation, miRNAs such as miR-7; miR-9;miR-9*; miR-25; miR-26a; miR-26b; miR-98; miR-124; miR-125b; miR-128;miR-132; miR-134; miR-138; miR-146; miR-182; miR-183; miR-200b;miR-200c; miR-213; miR-292-5p; miR-297; miR-322; miR-323-3p; miR-325;miR-337; miR-339; miR-345; miR-350; miR-351; miR-370; miR-425; miR-429;miR-433-5p; miR-446; miR-467; miR-874 (see Schratt et al., Nature439:283-289, 2006; Lugli et al., J Neurochem. 106:650-661, 2008; Bickerand Schratt, J Cell Mol Med. 12:1466-1476, 2008; Smalheiser and Lugli,Neuromolecular Med. 11:133-140, 2009; Rajasethupathy, Neuron,63:714-716, 2009; Kyc, RNA, 13:1224-1234, 2007; Yu, et al., Exp CellRes. 314:2618-2633, 2008; Cougot et al., J Neurosci. 28:13793-13804,2008; Kawahara, Brain Nerve, 60:1437-1444, 2008), and other small RNAssuch as BC200 RNA (Brain Cytoplasmic RNA 200-nucleotides; Dahm et al.,Seminars in Cell & Dev. Biol. 18: 216-223, 2007; Mus et al., Proc. Natl.Acad. Sci. USA., 104:10679-10684, 2007). Additional small RNAs useful inthe methods of the invention can be identified, for example, based ontheir enrichment in neurons (and in certain regions of the braindepending on a disease) and intracellular localization in axons and/ordendrites and/or spines and/or synapses. If urine samples are selectedfor conducting diagnostic methods of the invention, preferred small RNAsfor detection would be those small RNAs which are not significantlyexpressed in cells of the urinary system. Similarly, if blood samples(e.g., serum or plasma) are used for conducting diagnostic methods ofthe invention, preferred small RNAs for detection would be those smallRNAs which are not expressed or are present at very low levels in bloodcells.

The methods of the instant invention are based on measurement of levelsof certain small RNAs in bodily fluids. The use of bodily fluids thatcan be obtained by non-invasive or minimally invasive techniques (e.g.,as opposed to detection in the brain or CSF) allows for a cheap andminimally invasive or noninvasive diagnostic procedure. Preferred bodilyfluids for use in the methods of the invention are blood plasma, serum,urine, and saliva. However, any other bodily fluid can also be used.

Examples of useful methods for measuring small RNA level in bodilyfluids include hybridization with selective probes (e.g., using Northernblotting, bead-based flow-cytometry, oligonucleotide microchip[microarray], or solution hybridization assays such as Ambion mirVanamirna Detection Kit), polymerase chain reaction (PCR)-based detection(e.g., stem-loop reverse transcription-polymerase chain reaction[RT-PCR], quantitative RT-PCR based array method [qPCR-array]), ordirect sequencing by one of the next generation sequencing technologies(e.g., Helicos small RNA sequencing, miRNA BeadArray (Illumina), Roche454 (FLX-Titanium), and ABI SOLiD). For review of additional applicabletechniques see, e.g., Chen et al., BMC Genomics, 2009, 10:407; Kong etal., J Cell Physiol. 2009; 218:22-25.

In some embodiments, small RNAs are purified prior to quantification.Small RNAs (e.g., miRNAs) can be isolated and purified from bodilyfluids by various methods, including the use of commercial kits (e.g.,miRNeasy kit [Qiagen], MirVana RNA isolation kit [Ambion/ABI], miRACLE[Agilent], High Pure miRNA isolation kit [Roche], and miRNA Purificationkit [Norgen Biotek Corp.]), Trizol extraction (see Example 1, below),concentration and purification on anion-exchangers, magnetic beadscovered by RNA-binding substances, or adsorption of certain miRNA oncomplementary oligonucleotides.

In some embodiments, small RNA degradation in bodily fluid samplesand/or during small RNA purification is reduced or eliminated. Usefulmethods for reducing or eliminating small RNA degradation, include,without limitation, adding RNase inhibitors (e.g., RNasin Plus[Promega], SUPERase-In [ABI], etc.), use of guanidine chloride,guanidine isothiocyanate, N-lauroylsarcosine, sodium dodecyl sulphate(SDS), or a combination thereof. Also, when working with urine samples,lower risk of RNA degradation can be achieved when the sample has beenheld in the bladder for a shorter time (e.g., less than 4 hours).Reducing small RNA degradation in bodily fluid samples is particularlyimportant when sample storage and transportation is required prior tosmall RNA quantification.

To account for possible losses of a given small RNA during purification,potential RT-PCR inhibition, small RNA contaminants derived from dyingor damaged blood or urine cells during sample isolation and treatment,variations in kidney filtration, etc., various methods of experimentaldata normalization can be employed. For example, the followingnormalization methods can be used in the present invention:

a) Concentration of a target small RNA can be normalized to one ofubiquitous miRNAs (e.g., miR-16), small nucleolar RNAs (snoRNAs), miRNAswhich are not expressed in neurons (e.g., miR-122a, miR-10b, miR-141),U6 small nuclear RNA (U6 RNA), or neuron body miRNAs (e.g., miR-137,miR-181a, miR-491-5p, miR-298, miR-339 [Kye, RNA, 13:1224-1234, 2007],and others).

b) Synthetic small RNA (e.g., miRNA) oligonucleotides can be synthesizedand used as controls for losses during purification and RT-PCRinhibition (by adding them to bodily fluid samples before RNApurification).

c) To account for variations in kidney filtration (when working withurine samples), small RNA concentration in urine can be normalized oncreatinine and/or albumin level.

DEFINITIONS

The term “neuronal cell body” refers to the portion of a nerve cell thatcontains the nucleus surrounded by the cytoplasm and the plasma membranebut does not incorporate the dendrites or axons.

The term “neurite” as used herein refers to any projection from the cellbody of a neuron. This projection can be an axon, a dendrite, or aspine.

The term “axon” refers to a long, slender projection of a neuron thatconducts electrical impulses away from the neuron's cell body or soma.Axons are distinguished from dendrites by several features, includingshape (dendrites often taper while axons usually maintain a constantradius), length (dendrites are restricted to a small region around thecell body while axons can be much longer), and function (dendritesusually receive signals while axons usually transmit them). Axons anddendrites make contact with other cells (usually other neurons butsometimes muscle or gland cells) at junctions called synapses.

The term “dendrite” refers to a branched projection of a neuron thatacts to conduct the electrochemical stimulation received from otherneural cells to the cell body of the neuron from which the dendritesproject.

The terms “spine” or “dendritic spine” refer to a small membranousprotrusion from a neuron's dendrite that typically receives input from asingle synapse of an axon. Dendritic spines serve as a storage site forsynaptic strength and help transmit electrical signals to the neuronalcell body. Most spines have a bulbous head (the spine head), and a thinneck that connects the head of the spine to the shaft of the dendrite.The dendrites of a single neuron can contain hundreds to thousands ofspines. In addition to spines providing an anatomical substrate formemory storage and synaptic transmission, they may also serve toincrease the number of possible contacts between neurons.

The term “synapse” refers to specialized junctions, through whichneurons signal to each other and to non-neuronal cells such as those inmuscles or glands. A typical neuron gives rise to several thousandsynapses. Most synapses connect axons to dendrites, but there are alsoother types of connections, including axon-to-cell-body, axon-to-axon,and dendrite-to-dendrite. In the brain, each neuron forms synapses withmany others, and, likewise, each receives synaptic inputs from manyothers. As a result, the output of a neuron may depend on the input ofmany others, each of which may have a different degree of influence,depending on the strength of its synapse with that neuron. There are twomajor types of synapses, chemical synapses and electrical synapses. Inelectrical synapses, cells approach within about 3.5 nm of each other,rather than the 20 to 40 nm distance that separates cells at chemicalsynapses. In chemical synapses, the postsynaptic potential is caused bythe opening of ion channels by chemical transmitters, while inelectrical synapses it is caused by direct electrical coupling betweenboth neurons. Electrical synapses are therefore faster than chemicalsynapses.

Within the meaning of the present invention, the term “synapse and/orneurite small RNA” refers to small RNA (e.g., miRNA or BC200 RNA) which(i) is “neuron-enriched”, i.e., is present in increased amounts (e.g.,at least 5-times higher concentrations) in neurons, as compared to celltypes that can be a source of significant amounts of small RNA in abodily fluid being tested and (ii) is present in a synapse and/orneurite (i.e., axon and/or dendrite and/or spine). To be useful in thediagnostic methods of the present invention, such synapse and/or neuritesmall RNA should be detectable in bodily fluids as a result of itsrelease from neurons (e.g., due to neurite/synapse destruction orneuronal death).

The term “neuronal body small RNA” as used herein refers to small RNA(e.g., miRNA) which (i) is “neuron-enriched”, i.e., is present inincreased amounts (e.g., at least 5-times higher concentrations) inneurons, as compared to cell types that can be a source of significantamounts of small RNA in a bodily fluid being tested and (ii) is absentfrom or present in significantly lower concentrations in neurites orsynapses than in neuronal cell bodies.

The terms “neuronal pathology” and “pathological changes in neurons” areused herein to refer to metabolic and/or structural changes in neuronsassociated with neurite and/or synapse dysfunction and/or neuritedestruction and/or synapse loss.

The term “associated with” is used to encompass any correlation,co-occurrence and any cause-and-effect relationship.

The term “development of a neuronal pathology” is used herein to referto any negative change in the extent/severity of a metabolic and/orstructural change in individual neurons and/or any increase in thenumber of neurons affected. The phrase “improvement of a neuronalpathology” and similar terms refer to any positive change in theextent/severity of a metabolic and/or structural change in individualneurons and/or any decrease in the number of neurons affected.

As used herein, the term “small RNA” refers generally to a heterogeneousgroup of non-coding RNAs with a variety of regulatory functionsincluding chromatin architecture/epigenetic memory, transcription, RNAsplicing, RNA editing, mRNA translation, and RNA turnover. Thediagnostic methods of the present invention rely on detecting neuriteand/or synapse small RNAs, which can be detected in bodily fluids, suchas, for example, microRNAs (miRNAs), Brain Cytoplasmic RNAs BC1/BC200,etc. There are other classes of less characterized small RNAs which canbe also useful in the methods of the present invention (reviewed in Kim,Mol. Cells, 2005, 19: 1-15).

The terms “microRNA” or “miRNA” as used herein refer to a class of smallapproximately 22 nt long non-coding mature RNA molecules. They playimportant roles in the regulation of target genes by binding tocomplementary regions of messenger transcripts (mRNA) to repress theirtranslation or regulate degradation (Griffiths-Jones Nucleic AcidsResearch, 2006, 34, Database issue: D140-D144). Frequently, one miRNAcan target multiple mRNAs and one mRNA can be regulated by multiplemiRNAs targeting different regions of the 3′ UTR. Once bound to an mRNA,miRNA can modulate gene expression and protein production by affecting,e.g., mRNA translation and stability (Baek et al., Nature 455(7209):64(2008); Selbach et al., Nature 455(7209):58 (2008); Ambros, 2004,Nature, 431, 350-355; Bartel, 2004, Cell, 116, 281-297; Cullen, 2004,Virus Research., 102, 3-9; He et al., 2004, Nat. Rev. Genet., 5,522-531; and Ying et al., 2004, Gene, 342, 25-28). Examples of neuriteand/or synapse miRNAs useful in the methods of the present inventioninclude, without limitation, miR-7, miR-9, miR-9*, miR-25, miR-26a,miR-26b, miR-98, miR-124, miR-125b, miR-128, miR-132, miR-134, miR-138,miR-146, miR-182, miR-183, miR-200b, miR-200c, miR-213, miR-292-5p,miR-297, miR-322, miR-323-3p, miR-325, miR-337, miR-339, miR-345,miR-350, miR-351, miR-370, miR-425, miR-429, miR-433-5p, miR-446,miR-467 and miR-874. Information on most currently known miRNAs can befound in the miRNA database miRBase (available at the world wide web atmirbase.org). See also Burside et al., BMC Genomics 9:185 (2008);Williams et al., BMC Genomics 8:172 (2007); Landgraf et al., Cell129:1401 (2007).

The term “miRNA array” refers to a multiplex technology used inmolecular biology and in medicine. It consists of an arrayed series ofmultiple (e.g., thousands) microscopic spots of oligonucleotides, eachcontaining a specific sequence (probe) complementary to a particulartarget miRNA. After probe-target hybridization under high-stringencyconditions the resulting hybrids are usually detected and quantified byquantifying fluorophore-, silver-, or chemiluminescence-labeled targetsto determine relative abundance of miRNA. In the methods of the presentinvention, both custom-made and commercially available miRNA arrays canbe used. Examples of useful commercially available miRNA arrays (basedon various methods of target labeling, hybrid detection and analysis)include arrays produced by Agilent, Illumina, Invitrogen, Febit, and LCSciences.

The term “next generation sequencing technologies” broadly refers tosequencing methods which generate multiple sequencing reactions inparallel. This allows vastly increased throughput and yield of data.Non-limiting examples of commonly used next generation sequencingplatforms include Helicos small RNA sequencing, miRNA BeadArray(Illumina), Roche 454 (FLX-Titanium), and ABI SOLiD.

An “individual” or “subject” or “animal”, as used herein, refers tohumans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs,etc.) and experimental animal models of neurodegenerative diseases orother neuronal pathologies (see Examples, below). In a preferredembodiment, the subject is a human.

The term “urinary tract” refers to the organs and ducts, whichparticipate in the secretion and elimination of urine from the body.

The term “purified” as used herein refers to material that has beenisolated under conditions that reduce or eliminate the presence ofunrelated materials, i.e., contaminants, including native materials fromwhich the material is obtained. For example, RNA purification includeselimination of proteins, lipids, salts and other unrelated compoundspresent in bodily fluids. Besides, for some methods of analysis apurified miRNA is preferably substantially free of other RNAoligonucleotides contained in bodily fluid samples (e.g., rRNA and mRNAfragments, ubiquitous miRNAs, which are expressed at high levels inalmost all tissues [e.g., miR-16], etc.). As used herein, the term“substantially free” is used operationally, in the context of analyticaltesting of the material. Preferably, purified material substantiallyfree of contaminants is at least 50% pure; more preferably, at least 90%pure, and still more preferably at least 99% pure. Purity can beevaluated by chromatography, gel electrophoresis, composition analysis,biological assay, and other methods known in the art.

As used herein, the term “similarly processed” refers to samples (e.g.,bodily fluid samples or purified RNAs) which have been obtained usingthe same protocol.

The term “a control level” as used herein encompasses predeterminedstandards (e.g., a published value in a reference) as well as levelsdetermined experimentally in similarly processed samples from controlsubjects (e.g., age-matched healthy subjects, placebo treated patients,etc.).

The term “about” or “approximately” means within a statisticallymeaningful range of a value. Such a range can be within an order ofmagnitude, preferably within 50%, more preferably within 20%, still morepreferably within 10%, and even more preferably within 5% of a givenvalue or range. The allowable variation encompassed by the term “about”or “approximately” depends on the particular system under study, and canbe readily appreciated by one of ordinary skill in the art.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition. Cold SpringHarbor, N.Y.: Cold Spring Harbor Laboratory Press, 1989 (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds.(1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins,eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)];Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, APractical Guide To Molecular Cloning (1984); Ausubel, F. M. et al.(eds.). Current Protocols in Molecular Biology. John Wiley & Sons, Inc.,1994. These techniques include site directed mutagenesis as described inKunkel, Proc. Natl. Acad. Sci. USA 82: 488-492 (1985), U.S. Pat. No.5,071,743, Fukuoka et al., Biochem. Biophys. Res. Commun. 263: 357-360(1999); Kim and Maas, BioTech. 28: 196-198 (2000); Parikh andGuengerich, BioTech. 24: 4 28-431 (1998); Ray and Nickoloff, BioTech.13: 342-346 (1992); Wang et al., BioTech. 19: 556-559 (1995); Wang andMalcolm, BioTech. 26: 680-682 (1999); Xu and Gong, BioTech. 26: 639-641(1999), U.S. Pat. Nos. 5,789,166 and 5,932,419, Hogrefe, Strategies 14.3: 74-75 (2001), U.S. Pat. Nos. 5,702,931, 5,780,270, and 6,242,222,Angag and Schutz, Biotech. 30: 486-488 (2001), Wang and Wilkinson,Biotech. 29: 976-978 (2000), Kang et al., Biotech. 20: 44-46 (1996),Ogel and McPherson, Protein Engineer. 5: 467-468 (1992), Kirsch andJoly, Nuc. Acids. Res. 26: 1848-1850 (1998), Rhem and Hancock, J.Bacteriol. 178: 3346-3349 (1996), Boles and Miogsa, Curr. Genet. 28:197-198 (1995), Barrenttino et al., Nuc. Acids. Res. 22: 541-542 (1993),Tessier and Thomas, Meths. Molec. Biol. 57: 229-237, and Pons et al.,Meth. Molec. Biol. 67: 209-218.

EXAMPLES

The present invention is also described and demonstrated by way of thefollowing examples. However, the use of these and other examplesanywhere in the specification is illustrative only and in no way limitsthe scope and meaning of the invention or of any exemplified term.Likewise, the invention is not limited to any particular preferredembodiments described here. Indeed, many modifications and variations ofthe invention may be apparent to those skilled in the art upon readingthis specification, and such variations can be made without departingfrom the invention in spirit or in scope. The invention is therefore tobe limited only by the terms of the appended claims along with the fullscope of equivalents to which those claims are entitled.

Example 1 Comparison of Different Methods Used for miRNA Purificationfrom Serum or Plasma

Since most of the commercially available kits for miRNA isolation havebeen developed for miRNA purification from cells and tissues variouskits are compared with in-house modifications to adjust them for miRNAisolation from serum or plasma. Commercial kits include the miRNeasy kit(Qiagen), the MirVana RNA isolation kit (Ambion/ABI), miRACLE (Agilent),High Pure miRNA isolation kit (Roche), and miRNA Purification kit(Norgen Biotek Corp.). Besides, the in-house techniques based on the useof Trizol (Invitrogen) are developed. In some experiments, miRNA ispre-adsorbed on anion-exchangers, such as Q-Sepharose, or on magneticbeads covered with a RNA-binding material (Q-Sepharose (GE Healthcare),PEI-polyethyleneimine, or other). After Trizol deproteinization, RNA isprecipitated with isopropyl alcohol or additionally purified on silicacolumns. In some experiments, purified RNA is treated with RNAse-freeDNAse (Qiagen, ABI, Invitrogen or other). miRNA preparations obtained bydifferent methods are compared using RT PCR. The quality of miRNApreparations is also evaluated by measurement of the RT PCR inhibition(see Example 3, below).

miRNA was purified from plasma and serum samples obtained from the same5 healthy donors. 10⁷ copies of Arabidopsis thaliana miR-159a(ath-miR-159a) were spiked per 1 ml plasma or serum after addition ofguanidine-containing solution for evaluation of miRNA yield. Twotechniques, one based on MirVana Paris kit (Ambion/ABI), and anotherbased on Trizol (Invitrogen) deproteinization, and subsequentpurification on silica columns, were compared. After RNA purificationconcentrations of spiked miRNA and human endogenous miR-9, miR-16, andmiR-134 in final preps were measured by RT-PCR. MirVana Paris kit wasmore effective in miRNA isolation then the Trizol-based technique andwas selected for future experiments. Although all analyzed miRNA weredetectable in serum and plasma and both sample types are suitable formiRNA testing, the final PCR Ct values were about 2 cycles lower forplasma, and the latter was used in subsequent experiments. Based on thequantitative measurement of spiked ath-miR-159a, average yield of miRNAisolated from plasma with MirVana kit was 71.4%.

Example 2 Selection of miRNA for testing

Tested miRNAs were initially selected based on literature data on theirenrichment in brain compartments and presence in neurites (i.e., axonsand/or dendrites and/or spines) and/or synapses (Hua et al., BMCGenomics 2009, 10:214; Liang et al., BMC Genomics. 2007, 8:166; Landgrafet al., Cell. 2007, 129:1401-1414; Lee et al., RNA. 2008, 14:35-42;Schratt et al., Nature. 439:283-289, 2006; Lugli et al., J Neurochem.106:650-661, 2008; Bicker and Schratt, J Cell Mol Med., 12:1466-1476,2008; Smalheiser and Lugli, Neuromolecular Med. 11:133-140, 2009;Rajasethupathy, Neuron. 63:714-716, 2009; Kye, RNA 13:1224-1234, 2007;Yu et al., Exp Cell Res. 314:2618-2633, 2008; Cougot, et al., JNeurosci. 28:13793-13804, 2008; Kawahara, Brain Nerve. 60:1437-1444,2008; Schratt G. Rev Neurosci. 2009; 10:842-849) as well as on theirinvolvement in neurite- and synapse-associated processes (ThemiR-Ontology Data Base: available at the world wide web atferrolab.dmi.unict.it/miro/). For normalization, in addition to spikedmiRNA, ubiquitous miRNA, such as miR-16, and miRNA expressed in numeroustissues but not in brain, such as miR-10b and miR-141, were used.

Example 3 Detection of an Increase in Levels of Synapse and/or NeuritemiRNA in Plasma of AD Patients

Plasma samples were obtained from patients diagnosed with developed ADby cognitive test and brain imaging. Profiles of several neuron-enrichedmiRNAs from plasma of these patients were analyzed using RT-PCR withprimers and probes for each individual miRNA (ABI). The amount of RNAequivalent to 30 μL plasma were taken in each PCR reaction, and 1/15 ofRT product was taken into final PCR. Thus, the amount of miRNAequivalent to 2 μL plasma was detected. The results were normalized pervarious miRNA, usually per ubiquitous miR-16, converted into RelativeQuantity (RQ) of miRNA according the ABI protocol (2^(−ΔCt)), andcompared with miRNA profiles from age-matched controls. In addition,data obtained with neurite and/or synapse miRNA were compared with dataobtained with neuronal body miRNA.

As shown in FIGS. 1A-G, the data obtained clearly demonstrate thatconcentrations of many neuron-enriched miRNAs increase in plasma of ADpatients. However, this effect is much more prominent for neurite and/orsynapse miRNAs (miR-7 (A), miR-125b (B), miR-128 (C), miR-132 (D), andmiR-323-3p (E)) than for neuronal body miRNAs (miR-181a (F) andmiR-491-5p (G)).

Other techniques can be used for measuring miRNA concentration in bodilyfluids with some precautions. For example, application of nextgeneration sequencing technologies to quantitative analysis of miRNAsand other small RNAs in bodily fluids is complicated by two factors.First, fragments of ribosomal RNA (rRNA) and to a lesser degreemessenger RNA (mRNA) comprise major part of small oligonucleotidespresent in bodily fluids, which complicates sequencing of miRNAs andsome of the other small RNAs, which are present in a much smaller numberof copies. Second, some ubiquitous miRNAs, which are expressed at highlevels in almost all tissues (e.g., miR-16), can be present in bodilyfluids in the million times larger number of copies than miRNAs ofinterest. To overcome these problems, prior to performing quantitativesequencing of relatively rare neurite and/or synapse miRNAs and otherneurite and/or synapse small RNAs, the preparations of RNA from bodilyfluids can be depleted from rRNA fragments using, for example, SelectiveHybridization and Removal of rRNA kit (Invitrogen), and otheroligonucleotides present in a huge number of copies can be removed byhybridization with respective complementary DNA sequences. Thesedepleted RNA preparations can be then analyzed using one of newgeneration sequencing techniques, such as, e.g., Helicos small RNAsequencing or the miRNA BeadArray (Illumina). miRNAs and/or other smallRNAs, which provide the most reproducible and reliable results (i.e.,change in level characteristic of a certain neurodegenerative disease),can be selected as potential biomarkers and analyzed by RT-PCR or othermethods.

Example 4 Demonstration that the Increase in Levels of Neurite and/orSynapse miRNAs in MCI Patients is More Significant and Precedes theIncrease in Levels of Neuronal Body miRNAs

Plasma samples were obtained from patients diagnosed with MCI. Profilesof neuron-enriched miRNAs from plasma of these patients were analyzedusing RT-PCR with primers and probes for each individual miRNA (ABI).The amount of RNA equivalent to 30 μL plasma were taken in each PCRreaction, and 1/15 of RT product was taken into final PCR. Thus, theamount of miRNA equivalent to 2 μL plasma was detected. The results werenormalized per various miRNA, converted into Relative Quantity (RQ) ofmiRNA according the ABI protocol (2^(−Δct)), and compared with miRNAprofiles from age-matched controls.

When normalization per spiked non-human miRNA was performed, which givesrelative miRNA concentration per 1 ml plasma, some plasma samples fromMCI patients contained more neurite and/or synapse miRNAs (FIG. 2, miR-7(A) and miR-874 (B)).

At the same time concentrations of neuronal body miRNAs were not changedin the plasma of MCI patients (FIG. 2, miR-181a (C) and miR-491-5p (D)).

Similar results were obtained, when miRNA concentrations in plasma werenormalized per miR-141, which is expressed in many organs but not in thebrain (FIGS. 3A-B).

Example 5 Comparison of Neuron-Enriched miRNA Levels in Plasma ofControl, MCI and AD Patients

Since concentrations of neurite and/or synapse miRNAs increase andconcentrations of neuronal body miRNAs are practically unchanged inplasma of MCI patients, their ratios were used for analysis of diseasedevelopment. Plasma samples were obtained from patients diagnosed withMCI and AD. Profiles of neuron-enriched miRNA from plasma of thesepatients were analyzed using RT-PCR with primers and probes for eachindividual miRNA (ABI). The amount of RNA equivalent to 30 μL plasmawere taken in each PCR reaction, and 1/15 of RT product was taken intofinal PCR. Thus, the amount of miRNA equivalent to 2 μL plasma wasdetected. Then the concentrations of neurite and/or synapse miRNAs werenormalized per miRNA, located mainly in neuronal body, according the ABIprotocol (2^(−Δct)), and compared with respective numbers fromage-matched controls.

As shown in FIGS. 4A-D, the data obtained demonstrate a clear trend ofincreasing concentrations of some neurite and/or synapse miRNAs (i.e.,miR-128 (A), miR-132 (B), miR-370 (C), and miR-125b (D)) from Control toMCI to AD. These data suggest that periodic screening of elderly peoplecan help with early diagnostics and monitoring of MCI and AD.

Example 6 Detection of Neurite Destruction and Synapse Loss (in theAbsence of Massive Neuronal Cell Death) in Animal Models of Early andMild AD by Analysis of Neurite and/or Synapse miRNAs in Blood

The following animal models of AD can be used to detect neuritedestruction and/or synapse loss (in the absence of massive neuronal celldeath) using the analysis of neurite and/or synapse miRNAs in bodilyfluids. The same animal models are useful for testing the sensitivityand adjusting the conditions of the diagnostic and monitoring methods ofthe present invention and for identifying additional neurite and/orsynapse miRNAs and other small RNA molecules that can be used as markersof neurodegenerative diseases.

Various transgenic mice models are currently available that overexpressFamilial Alzheimer's disease (FAD) mutant forms of human APP. Mostcurrently studied models show cognitive deficits and age-relateddisruption of synaptic markers and amyloid plaque deposition, but fewstrains show evidence of significant cell death (Janus et al. 2000; Ashe2001; Chapman et al. 2001; Richardson & Burns 2002). Examples of suchtransgenic mice are (i) PDAPP mice overexpressing hAPP V717F, (ii)Tg2576 mice overexpressing hAPP 695 mutated with both K670N and M671L(Hsiao et al., 1996), (iii) TgAPP/Ld/2 mice overexpressing hAPP V6421;(iv) mice overexpressing hAPP V717I; (v) human APP transgenic mice withmutation of Asp-664, which prevents caspase cleavage and accumulation ofcytotoxic peptide APP-C31 with partial reversal of Alzheimer's-likepathology (Galvan et al. Proc Natl Acad Sci USA. 2006; 103:7130-7135).Also useful is a double mutant transgenic mouse model expressing APPminigenes that encode FAD-linked APP mutants and an early-onset familialAD (FAD)-linked human presenilin 1 (PS1) variant (A246E) and a chimericmouse/human APP harboring mutations linked to Swedish FAD kindreds(APPswe) (see U.S. Pat. No. 5,912,410; Borchelt et al., Neuron 1997,19:939-945; Holcomb et al., 1998). These mice develop numerous amyloiddeposits much earlier than age-matched mice expressing APPswe andwild-type human PS1. Expression of APP minigenes that encode FAD-linkedAPP mutants and, in particular, co-expression of the mutant human PS1A246E and APPswe elevates levels of AP in the brain, and these micedevelop numerous diffuse Aβ deposits and plaques in the hippocampus andcortex (Calhoun et al., Proc. Natl. Acad. Sci. USA 1999;96:14088-14093). Similarly to humans suffering from AD, these and othertransgenic animal models are characterized by various cognitive defectssuch as loss of neurons, learning deficits, problems in objectrecognition memory, and problems with alternation-spatial reference andworking memory (Chen et al., Nature 2000; 408:975-979).

To detect neurite destruction and synapse loss (in the absence ofmassive neuronal cell death), neurite and/or synapse miRNAs are isolatedfrom the blood serum/plasma of AD model transgenic mice and analyzed byRT-PCR, and data obtained are compared with brain histopathology.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description. Suchmodifications are intended to fall within the scope of the appendedclaims.

All patents, applications, publications, test methods, literature, andother materials cited herein are hereby incorporated by reference intheir entirety as if physically present in this specification.

1-6. (canceled)
 7. A method for diagnosing Alzheimer's disease (AD) in asubject, which comprises: a. determining the level of at least onesynapse or neurite small RNA in a bodily fluid sample from the subject;b. comparing the level of the small RNA in the bodily fluid sample fromthe subject with a control level of the small RNA, and c. (i)identifying the subject as being afflicted with AD when the level of thesmall RNA in the bodily fluid sample from the subject is increased ascompared to the control or (ii) identifying the subject as not beingafflicted with AD when the level of the small RNA in the bodily fluidsample from the subject is not increased as compared to the control. 8.The method of claim 7, wherein AD is diagnosed prior to massive neuronalcell death characteristic of AD. 9-13. (canceled)
 14. The method ofclaim 7, wherein the small RNA is miRNA.
 15. The method of claim 14,wherein the miRNA is a member selected from the group consisting ofmiR-7, miR-9, miR-9*, miR-25, miR-26a, miR-26b, miR-98, miR-124,miR-125b, miR-128, miR-132, miR-134, miR-138, miR-146, miR-182, miR-183,miR-200b, miR-200c, miR-213, miR-292-5p, miR-297, miR-322, miR-323-3p,miR-325, miR-337, miR-339, miR-345, miR-350, miR-351, miR-370, miR-425,miR-429, miR-433-5p, miR-446, miR-467, and miR-874.
 16. (canceled) 17.The method of claim 7, wherein the control level of the small RNA is thelevel of said small RNA in a similarly processed bodily fluid samplefrom an age-matched control subject.
 18. The method of claim 7, whereinthe control level of the small RNA is the level of said small RNA in asimilarly processed bodily fluid sample from the same subject obtainedin the past.
 19. The method of claim 7, wherein the control level of thesmall RNA is a predetermined standard. 20-23. (canceled)
 24. The methodof claim 7, wherein the bodily fluid sample is blood plasma. 25.(canceled)
 26. The method of claim 7, wherein, prior to step (a), thesmall RNA is purified from the bodily fluid sample. 27-30. (canceled)31. A method for monitoring the effectiveness of a treatment of aneuronal pathology in a subject, which comprises: a. determining thelevel of at least one synapse or neurite small RNA in a bodily fluidsample from the subject obtained prior to initiation of the treatment;b. determining the level of the small RNA in one or more bodily fluidsample(s) from the subject obtained in the course of or following thetreatment, and c. comparing the level of the small RNA determined insteps (a) and (b), and optionally between different samples in step (b).32. The method of claim 31, which further comprises (d) (i) determiningthat the treatment is effective if the level of the small RNA hasdecreased in the course of or following the treatment or (ii)determining that the treatment is not effective if the level of thesmall RNA has not decreased in the course of or following the treatment.33-37. (canceled)
 38. The method of claim 31, wherein the small RNA ismiRNA.
 39. The method of claim 38, wherein the miRNA is a memberselected from the group consisting of miR-7, miR-9, miR-9*, miR-25,miR-26a, miR-26b, miR-98, miR-124, miR-125b, miR-128, miR-132, miR-134,miR-138, miR-146, miR-182, miR-183, miR-200b, miR-200c, miR-213,miR-292-5p, miR-297, miR-322, miR-323-3p, miR-325, miR-337, miR-339,miR-345, miR-350, miR-351, miR-370, miR-425, miR-429, miR-433-5p,miR-446, miR-467, and miR-874. 40-48. (canceled)
 49. A tool fordetecting neurite destruction and synapse loss, associated with aneuronal pathology, or for monitoring a neuronal pathology or formonitoring the effectiveness of a treatment of a neuronal pathologycomprising primers and/or probes for one or more miRNA selected from thegroup consisting of miR-7, miR-9, miR-9*, miR-25, miR-26a, miR-26b,miR-98, miR-124, miR-125b, miR-128, miR-132, miR-134, miR-138, miR-146,miR-182, miR-183, miR-200b, miR-200c, miR-213, miR-292-5p, miR-297,miR-322, miR-323-3p, miR-325, miR-337, miR-339, miR-345, miR-350,miR-351, miR-370, miR-425, miR-429, miR-433-5p, miR-446, miR-467, andmiR-874, and further comprising primers and/or probes for one or moremiRNA selected from the group consisting of miR-181a, miR-491-5p,miR-10b, and miR-141.